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PubMed2003

Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments.

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.

Base Sequence↗
PubMed1999

Isolation, cloning, and expression of an acid phosphatase containing phosphotyrosyl phosphatase activity from Prevotella intermedia.

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an M(r) of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of Morganella morganii, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.

Acid Phosphatase↗
PubMed1986

Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.

The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.

Animals↗
PubMed2001

Mutational analysis of the PRL receptor gene in human breast tumors with differential PRL receptor protein expression.

PRL is a major growth and differentiating hormone in the human breast, with activation of the PRL-PRL receptor complex increasingly recognized as an important mechanism in the induction and progression of mammary tumors. Although constitutive activation of various hormone and growth factor receptors is newly recognized as a common cause of tumor development, the PRL receptor gene has not been analyzed for similar aberrations in breast and other tumors. Therefore, using bacterial artificial chromosomes containing the PRL receptor gene and intron-spanning PCR, we determined the exon-surrounding intron sequences providing primers for the first analysis of the entire coding region of the human PRL receptor gene. We examined the presence of PRL receptor in 41 breast tumors by immunohistochemistry and attempted a correlation of its expression to pathological grading of the disease. Then tumor cells were isolated by laser capture microdissection to examine DNA from 30 patients for PRL receptor mutations. The PRL receptor immunoreactive score did not correlate to the tumor size, histopathological grading, age, or family history of patients. PRL receptor immunoreactivity was predominantly found in steroid hormone receptor-positive tumors, but without overall correlation of immunoreactive score. In both PRL receptor-positive and PRL receptor- negative breast cancer cells, direct sequencing of the coding sequence of the PRL receptor gene did not detect any somatic or hereditary gene aberrations. In conclusion, PRL receptor mutations do not appear to be common in human breast cancer, suggesting that constitutive activation of the PRL receptor can be excluded as a major cause of mammary tumor genesis. The molecular structure of the PRL receptor seems to remain intact in tumor tissue, and systemic and local production of PRL may participate in tumor cell growth and proliferation through functional receptors.

Adult↗
PubMed1996

Expression of the major inner capsid protein of the epizootic haemorrhagic disease virus in baculovirus and potential diagnostic use.

The RNA 7 encoding the major capsid protein (VP7) of epizootic haemorrhagic disease virus (EHDV), Australian serotype 2 (strain CS439), was cloned and the complete nucleotide sequence was determined. The coding region contained 1047 nucleotides (nt) capable of encoding a predicted 349 amino acid (aa) polypeptide with a calculated molecular size of 38.087 kDa. When the VP7 gene was expressed in bacterial or yeast expression systems, the expression product showed weak or no reactivity with polyclonal antibodies to EHDV. Therefore, the expression of the VP7 gene in baculovirus was pursued. The expressed EHDV VP7 was similar in antigenicity to that of the native virus as revealed by its reactivity in ELISA with monoclonal antibody (MAb) specific to EHDV. Preliminary ELISA results indicated that the recombinant protein binds to EHDV antibodies in serum and that these antibodies block the binding of EHDV-specific MAb. The availability of a reliable EHDV recombinant VP7 could enhance our existing assay for detection of EHDV-specific antibodies.

Amino Acid Sequence↗
PubMed2006

High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study.

Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

Bacterial Typing Techniques↗
PubMed1984

Developmentally regulated expression of chimeric genes containing muscle actin DNA sequences in transfected myogenic cells.

A recombinant plasmid containing 2/3 of the rat skeletal muscle actin structural gene plus 730 bp of its 5' flanking region, spliced to the 3' end of the human epsilon-globin gene, was introduced into cells of the rat myogenic line L8. Myogenic clones carrying the actin/globin chimeric gene were isolated. In many of these clones, the expression of the gene greatly increased during differentiation (up to greater than 50-fold) and, in some clones, the amount of the chimeric gene transcripts in the differentiated cultures exceeded that of the native muscle actin gene transcripts. Furthermore, the temporal relation between differentiation of the cultures and the accumulation of the transcripts from the transferred genes was very similar to that of the native skeletal muscle actin gene, suggesting a similar mechanism of regulation. Endonuclease S1 analysis indicated a correct initiation and termination of the mRNA but suggested that a fraction of the chimeric actin/globin transcripts was not properly processed. To test whether the increased expression of the transferred gene which occurred during differentiation was determined by DNA sequences in the 5' region of the muscle actin gene, a plasmid (p alpha-CAT) containing 730 bp of the 5' flanking region of the rat skeletal muscle actin gene (plus the exon of the 5' untranslated region, and 25 bp of the first intron), spliced to the bacterial structural gene coding for chloramphenicol acetyl transferase (CAT), was constructed and introduced into L8 cells. In the majority of the isolated clones containing this plasmid, CAT activity increased many-fold during differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1985

Comparative studies on the structural gene for the ribosomal protein S1 in ten bacterial species.

By applying the Southern blot technique we compared the structural gene rpsA for ribosomal protein S1 and its preceding sequence from Escherichia coli with nine other bacterial species. We found high homology among the structural genes of E. coli and other gram-negative but not gram-positive bacteria. In contrast, the regulatory sequence preceding the structural gene was not highly conserved among the organisms studied. Cloning and DNA sequence analysis of a 1.2 kb fragment coding for most of the structural gene for S1 from Providencia localized some strongly conserved parts of the DNA sequence, despite the fact that the codon usage showed considerable divergence from that of E. coli.

Amino Acid Sequence↗
PubMed1996

Identification of cis-acting DNA sequences involved in the transcription of the virulence regulatory gene spvR in Salmonella typhimurium.

The SpvR protein is a DNA-binding protein of the LysR family, required for the transcription of the spvABCD virulence operon of Salmonella typhimurium. An alternative sigma factor, sigma S (RpoS), in conjunction with SpvR, controls the transcription of the spvR gene. In this study, we used a combination of primer extension experiments and deletion/fusion analyses of the spvR gene to identify sequences involved in spvR transcription in S. typhimurium. When induced in the stationary phase of growth in rich medium or during carbon starvation, transcription of spvR in S. typhimurium is driven by a single promoter (spvRp1) and initiates 17 nucleotides upstream of the spvR start codon. The level of spvR transcription originating at spvRp1 was 20-fold higher in the wild-type strain than in the rpoS mutant. In both strains, however, transcription at spvRp1 requires the SpvR protein. 5' Deletions up to position -86, relative to the spvR start codon, did not inhibit inducibility by sigma S and/or SPVR. In contrast, 5' deletion up to -75 abolished the activation of spvRp1 by SpvR in both the wild-type strain and rpoS mutant. Within the 11-bp sequence lying between position -86 and position -75, a 10-bp consensus motif TNTNTGCANA, present in both the spvR and spvA promoter regions, was identified and may contain the DNA recognition site for SpvR. In addition, we detected initiation of transcription within the spvR coding region. This finding may have implications for comparative studies of regulation with spvR gene fusions.

Bacterial Proteins↗
PubMed1996

Frequent microsatellite instabilities and analyses of the related genes in familial gastric cancers.

Microsatellite instability or replication error seems to be related to defective DNA mismatch repair genes, such as hMSH2, hMLH1, hPMS1 and hPMS2, which have been identified as causative genes of hereditary nonpolyposis colorectal cancers (HNPCC). Recently, it was reported that mutations at the simple repeated sequences in the transforming growth factor-beta type II receptor (TGF-beta RII) gene occurred in replication error-positive colorectal cancers. To determine genetic alterations in familial gastric cancers (FGC, we examined replication error using eight microsatellite DNA markers, and screened mutations in the hMSH2, hMLH1 and TGF-beta RII genes in six cases from four FGC kindreds. Moreover, hMTH1, a human homolog of the bacterial mutT gene, was also screened. Four of six (67%) cancers showed the replication error-positive phenotype, indicating that microsatellite instability is highly associated with not only HNPCC, but also FGC. No germline mutation was found in the whole coding sequences of hMSH2 and hMTH1, or in the conservative regions of hMLH1 in any patient, while one cancer DNA showed a somatic mutation at codon 682 (threonine to alanine) in hMSH2. No alteration was found at the small repeated sequences in TGF-beta RII in FGC tumor DNA. These results indicate that the carcinogenetic process of FGC may be different from that of HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed2004

A resource for large-scale RNA-interference-based screens in mammals.

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.

Animals↗
PubMed1998

Cloning of genes coding for the three subunits of thiocyanate hydrolase of Thiobacillus thioparus THI 115 and their evolutionary relationships to nitrile hydratase.

Thiocyanate hydrolase is a newly found enzyme from Thiobacillus thioparus THI 115 that converts thiocyanate to carbonyl sulfide and ammonia (Y. Katayama, Y. Narahara, Y. Inoue, F. Amano, T. Kanagawa, and H. Kuraishi, J. Biol. Chem. 267:9170-9175, 1992). We have cloned and sequenced the scn genes that encode the three subunits of the enzyme. The scnB, scnA, and scnC genes, arrayed in this order, contained open reading frames encoding sequences of 157, 126, and 243 amino acid residues, respectively, for the beta, alpha, and gamma subunits, respectively. Each open reading frame was preceded by a typical Shine-Dalgarno sequence. The deduced amino-terminal peptide sequences for the three subunits were in fair agreement with the chemically determined sequences. The protein molecular mass calculated for each subunit was compatible with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From a computer analysis, thiocyanate hydrolase showed significant homologies to bacterial nitrile hydratases known to convert nitrile to the corresponding amide, which is further hydrolyzed by amidase to form acid and ammonia. The two enzymes were homologous over regions corresponding to almost the entire coding regions of the genes: the beta and alpha subunits of thiocyanate hydrolase were homologous to the amino- and carboxyl-terminal halves of the beta subunit of nitrile hydratase, and the gamma subunit of thiocyanate hydrolase was homologous to the alpha subunit of nitrile hydratase. Comparisons of the catalytic properties of the two homologous enzymes support the model for the reaction steps of thiocyanate hydrolase that was previously presented on the basis of biochemical analyses.

Amino Acid Sequence↗
PubMed2002

The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis.

As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource. To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations. Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced. This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring co-linearity of the eventual sequence assembly with the actual genome. A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.

Cloning, Molecular↗
PubMed1999

Substrate binding and catalysis by ribonuclease P from cyanobacteria and Escherichia coli are affected differently by the 3' terminal CCA in tRNA precursors.

We have studied the effect of the 3' terminal CCA sequence in precursors of tRNAs on catalysis by the RNase P RNA or the holoenzyme from the cyanobacterium Synechocystis sp. PCC 6803 in a completely homologous system. We have found that the absence of the 3' terminal CCA is not detrimental to activity, which is in sharp contrast to what is known in other bacterial systems. We have found that this is also true in other cyanobacteria. This situation correlates with the anomalous structure of the J15/16 loop in cyanobacteria, which is an important loop in the CCA interaction in Escherichia coli RNase P, and with the fact that cyanobacteria do not code the CCA sequence in the genome but add it posttranscriptionally. Modification of nucleotides 330-332 in the J15/16 loop of Synechocystis RNase P RNA from GGU to CCA has a modest effect on kcat for CCA-containing substrates and has no effect on cleavage-site selection. We have developed a direct physical assay of the interaction between RNase P RNA and its substrate, which was immobilized on a filter, and we have determined that Synechocystis RNase P RNA binds with better affinity the substrate lacking CCA than the substrate containing it. Our results indicate a mode of substrate binding in RNase P from cyanobacteria that is different from binding in other eubacteria and in which the 3' terminal CCA is not involved.

Base Sequence↗
PubMed2000

Visualization of a cytoskeleton-like FtsZ network in chloroplasts.

It has been a long-standing dogma in life sciences that only eukaryotic organisms possess a cytoskeleton. Recently, this belief was questioned by the finding that the bacterial cell division protein FtsZ resembles tubulin in sequence and structure and, thus, may be the progenitor of this major eukaryotic cytoskeletal element. Here, we report two nuclear-encoded plant ftsZ genes which are highly conserved in coding sequence and intron structure. Both their encoded proteins are imported into plastids and there, like in bacteria, they act on the division process in a dose-dependent manner. Whereas in bacteria FtsZ only transiently polymerizes to a ring-like structure, in chloroplasts we identified persistent, highly organized filamentous scaffolds that are most likely involved in the maintenance of plastid integrity and in plastid division. As these networks resemble the eukaryotic cytoskeleton in form and function, we suggest the term "plastoskeleton" for this newly described subcellular structure.

Arabidopsis↗
PubMed1991

Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes.

DNA sequences from specific genes, amplified by the polymerase chain reaction technique, were used as substrata for nonisotopic restriction endonuclease fragment length polymorphism differentiation of rickettsial species and genotypes. The products amplified using a single pair of oligonucleotide primers (derived from a rickettsial citrate synthase gene sequence) and cleaved with restriction endonucleases were used to differentiate almost all recognized species of rickettsiae. A second set of primers was used for differentiation of all recognized species of closely related spotted fever group rickettsiae. The procedure circumvents many technical obstacles previously associated with identification of rickettsial species. Multiple amplified DNA digest patterns were used to estimate the intraspecies nucleotide sequence divergence for the genes coding for rickettsial citrate synthase and a large antigen-coding gene of the spotted fever group rickettsiae. The estimated relationships deduced from these genotypic data correlate reasonably well with established rickettsial taxonomic schemes.

Antigens, Bacterial↗
PubMedDate not supplied

New insights on the comma-less theory.

The comma-less hypothesis represents a theoretical effort to describe one of the steps in the early evolution of the translation apparatus. This hypothesis emphasizes the advantages that a RNY coding pattern would have provided in a primitive RNA adaptor-catalyst system. This theory has been debated for years, both in conceptual and statistical terms, and no consensus about its validity has been ascertained. In this work, a statistical model refuting this theory was reconsidered. This new approach eliminates the bias due to the absence of stop codons in the open reading frame, and to the amino acid composition of bacterial genes. The results obtained support the biological significance of the RNY coding pattern.

Amino Acids↗